RESUMO
BACKGROUND: Breast cancer is the most common malignant tumor disease and the leading cause of female mortality. The evolution of nanomaterials science opens the opportunity to improve traditional cancer therapies, enhancing therapy efficiency and reducing side effects. METHODS AND MAJOR RESULTS: Herein, protein cages conceived as enzymatic nanoreactors were designed and produced by using virus-like nanoparticles (VLPs) from Brome mosaic virus (BMV) and containing the catalytic activity of glucose oxidase (GOx) enzyme. The GOx enzyme was encapsulated into the BMV capsid (VLP-GOx), and the resulting enzymatic nanoreactors were coated with human serum albumin (VLP-GOx@HSA) for breast tumor cell targeting. The effect of the synthesized GOx nanoreactors on breast tumor cell lines was studied in vitro. Both nanoreactor preparations VLP-GOx and VLP-GOx@HSA showed to be highly cytotoxic for breast tumor cell cultures. Cytotoxicity for human embryonic kidney cells was also found. The monitoring of nanoreactor treatment on triple-negative breast cancer cells showed an evident production of oxygen by the catalase antioxidant enzyme induced by the high production of hydrogen peroxide from GOx activity. CONCLUSIONS AND IMPLICATIONS: The nanoreactors containing GOx activity are entirely suitable for cytotoxicity generation in tumor cells. The HSA functionalization of the VLP-GOx nanoreactors, a strategy designed for selective cancer targeting, showed no improvement in the cytotoxic effect. The GOx containing enzymatic nanoreactors seems to be an interesting alternative to improve the current cancer therapy. In vivo studies are ongoing to reinforce the effectiveness of this treatment strategy.
RESUMO
Immunogenic cell death (ICD) is a form of cell death characterized by the release of danger signals required to trigger an adaptive immune response against tumor-associated antigens. Silver nanoparticles (AgNP) display anti-proliferative and cytotoxic effects in tumor cells, but it has not been previously studied whether AgNP act as an ICD inductor. The present study evaluated the in vitro release of calreticulin as a damage-associated molecular pattern (DAMP) associated with the cytotoxicity of AgNP and their in vivo anti-cancer effects. In vitro, mouse CT26 colon carcinoma and MCA205 fibrosarcoma cells were exposed to AgNP and then cell proliferation, adhesion, and release of calreticulin were determined. The results indicated there were time- and concentration-related anti-proliferative effects of AgNP in both the CT26 and MCA205 lines. Concurrently, changes in cell adhesion were detected mainly in the CT26 cells. Regarding DAMP detection, a significant increase in calreticulin was observed only in CT26 cells treated with doxorubicin and AgNP; however, no differences were found in the MCA205 cells. In vivo, the survival and growth of subcutaneous tumors were monitored after vaccination of mice with cell debris from tumor cells treated with AgNP or after intra-tumoral administration of AgNP to established tumors. Consequently, anti-tumoral prophylactic immunization with AgNP-dead cells failed to protect mice from tumor re-challenge; intra-tumor injection of AgNP did not induce a significant effect. In conclusion, there was a noticeable anti-tumoral effect of AgNP in vitro in both CT26 and MCA205 cell lines, accompanied by the release of calreticulin in CT26 cells. In vivo, immunization with cell debris derived from AgNP-treated tumor cells failed to induce a protective immune response in the cancer model mice. Clearly, further research is needed to determine if one could combine AgNP with other ICD inducers to improve the anti-tumor effect of these nanoparticles in vivo.
Assuntos
Antineoplásicos , Nanopartículas Metálicas , Camundongos , Animais , Calreticulina/metabolismo , Calreticulina/farmacologia , Prata , Morte Celular Imunogênica , Morte Celular , Antineoplásicos/uso terapêutico , Linhagem Celular TumoralRESUMO
Gaucher disease is a genetic disorder and the most common lysosomal disease caused by the deficiency of enzyme ß-glucocerebrosidase (GCase). Although enzyme replacement therapy (ERT) is successfully applied using mannose-exposed conjugated glucocerebrosidase, the lower stability of the enzyme in blood demands periodic intravenous administration that adds to the high cost of treatment. In this work, the enzyme ß-glucocerebrosidase was encapsulated inside virus-like nanoparticles (VLPs) from brome mosaic virus (BMV), and their surface was functionalized with mannose groups for targeting to macrophages. The VLP nanoreactors showed significant GCase catalytic activity. Moreover, the Michaelis-Menten constants for the free GCase enzyme (KM =0.29â mM) and the functionalized nanoreactors (KM =0.32â mM) were similar even after chemical modification. Importantly, the stability of enzymes under physiological conditions (pHâ 7.4, 37 °C) was enhanced by ≈11-fold after encapsulation; this is beneficial for obtaining a higher blood circulation half-life, which may decrease the cost of therapy by reducing the requirement of multiple intravenous injections. Finally, the mannose receptor targeted enzymatic nanoreactors showed enhanced internalization into macrophage cells. Thus, the catalytic activity and cell targeting suggest the potential of these nanoreactors in ERT of Gaucher's disease.
Assuntos
Doença de Gaucher , Terapia de Reposição de Enzimas , Doença de Gaucher/tratamento farmacológico , Doença de Gaucher/genética , Glucosilceramidase/genética , Humanos , Manose , NanotecnologiaRESUMO
Nanomaterials have become part of our daily lives, particularly nanoparticles contained in food, water, cosmetics, additives and textiles. Nanoparticles interact with organisms at the cellular level. The cell membrane is the first protective barrier against the potential toxic effect of nanoparticles. This first contact, including the interaction between the cell membranes -and associated proteins- and the nanoparticles is critically reviewed here. Nanoparticles, depending on their toxicity, can cause cellular physiology alterations, such as a disruption in cell signaling or changes in gene expression and they can trigger immune responses and even apoptosis. Additionally, the fundamental thermodynamics behind the nanoparticle-membrane and nanoparticle-proteins-membrane interactions are discussed. The analysis is intended to increase our insight into the mechanisms involved in these interactions. Finally, consequences are reviewed and discussed.
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Nanopartículas , Nanoestruturas , Membrana Celular , Cosméticos , TermodinâmicaRESUMO
Abstract 20. Conventional glass ionomer cements are used as dental provisional restorative materials, which present several advantages such as adhesion to the tooth mineral phase among others. On the other hand, the knowledge about biological property of glass ionomers shows various approaches and results. In this work, it was studied the in vitro biological response of human gingival fibroblasts in contact with commercial cements of glass ionomer: Mirafil® and Ionglass® and with their extracts, according to ISO 10993. The extracts of the cements, in which the cells were cultured, were adjusted at different concentrations ranging 0.1% to 100%. The cellular metabolic activity of gingival fibroblasts was measured using the Alamar Blue® reagent. The results showed a significant effect on the cellular metabolic activity correlated with the concentration of liberated ions (Al³+ and Ca²+) for both ionomers, as well as the pH variations of the culture media. This could mean that the cellular metabolic activity is substantially influenced by ions and pH of the cell culture.
Resumen 24. Los cementos de ionómero de vidrio convencionales se utilizan como materiales de restauración provisional para uso dental, los cuales presentan varias ventajas como la adhesión a la fase mineral de los dientes. Por otro lado, las propiedades biológicas de los ionómeros de vidrio muestran diversos enfoques y resultados. En éste trabajo se estudió la respuesta biológica in vitro de fibroblastos gingivales humanos en contacto con cementos comerciales de ionómero de vidrio: Mirafil® e Ionglass® y con sus respectivos extractos según la norma ISO 10993. Los extractos de los cementos en los que se cultivaron las células estaban en diferentes concentraciones: de 0.1% a 100%. La actividad metabólica celular se midió usando el reactivo Alamar Blue®. Los resultados mostraron un efecto significativo sobre la actividad metabólica celular correlacionada con la concentración de iones liberados (Al³+ y Ca²+) para ambos ionómeros, así como las variaciones de pH de los medios de cultivo. Ello podria explicar la influencia por los iones y el pH del cultivo celular en la actividad metabólica celular.
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Cemento Dentário , Restauração Dentária Temporária , Cimentos de Ionômeros de Vidro/análise , Sobrevivência Celular , ÍonsRESUMO
BACKGROUND: Tamoxifen is the standard endocrine therapy for breast cancers, which require metabolic activation by cytochrome P450 enzymes (CYP). However, the lower and variable concentrations of CYP activity at the tumor remain major bottlenecks for the efficient treatment, causing severe side-effects. Combination nanotherapy has gained much recent attention for cancer treatment as it reduces the drug-associated toxicity without affecting the therapeutic response. RESULTS: Here we show the modular design of P22 bacteriophage virus-like particles for nanoscale integration of virus-driven enzyme prodrug therapy and photodynamic therapy. These virus capsids carrying CYP activity at the core are decorated with photosensitizer and targeting moiety at the surface for effective combinatory treatment. The estradiol-functionalized nanoparticles are recognized and internalized into ER+ breast tumor cells increasing the intracellular CYP activity and showing the ability to produce reactive oxygen species (ROS) upon UV365 nm irradiation. The generated ROS in synergy with enzymatic activity drastically enhanced the tamoxifen sensitivity in vitro, strongly inhibiting tumor cells. CONCLUSIONS: This work clearly demonstrated that the targeted combinatory treatment using multifunctional biocatalytic P22 represents the effective nanotherapeutics for ER+ breast cancer.
Assuntos
Antineoplásicos Hormonais/administração & dosagem , Bacteriófago P22/enzimologia , Neoplasias da Mama/tratamento farmacológico , Sistema Enzimático do Citocromo P-450/administração & dosagem , Fármacos Fotossensibilizantes/administração & dosagem , Tamoxifeno/administração & dosagem , Antineoplásicos Hormonais/farmacologia , Bacteriófago P22/química , Biocatálise , Neoplasias da Mama/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/farmacologia , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Terapia Enzimática , Feminino , Humanos , Células MCF-7 , Modelos Moleculares , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de Estrogênio/metabolismo , Tamoxifeno/farmacologiaRESUMO
Bovine bone matrix Nukbone® (NKB) is an osseous tissue-engineering biomaterial that retains its mineral and organic phases and its natural bone topography and has been used as a xenoimplant for bone regeneration in clinics. There are not studies regarding its influence of the NKB in the behavior of cells during the repairing processes. The aim of this research is to demonstrate that NKB has an osteoinductive effect in human mesenchymal stem cells from amniotic membrane (AM-hMSCs). Results indicated that NKB favors the AM-hMSCs adhesion and proliferation up to 7 days in culture as shown by the scanning electron microscopy and proliferation measures using an alamarBlue assay. Furthermore, as demonstrated by reverse transcriptase polymerase chain reaction, it was detected that two gene expression markers of osteoblastic differentiation: the core binding factor and osteocalcin were higher for AM-hMSCs co-cultured with NKB in comparison with cultivated cells in absence of the biomaterial. As the results indicate, NKB possess the capability for inducing successfully the osteoblastic differentiation of AM-hMSC, so that, NKB is an excellent xenoimplant option for repairing bone tissue defects.
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Diferenciação Celular , Proliferação de Células , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Alicerces Teciduais , Âmnio , Animais , Bovinos , Humanos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
When human tooth enamel is observed with the Transmission Electron Microscope (TEM), a structural defect is registered in the central region of their nanometric grains or crystallites. This defect has been named as Central Dark Line (CDL) and its structure and function in the enamel structure have been unknown yet. In this work we present the TEM analysis to these crystallites using the High Angle Annular Dark Field (HAADF) technique. Our results suggest that the CDL region is the calcium richest part of the human tooth enamel crystallites.